Genomic structure of the sandworm, Perinereis vancaurica tetradentata, troponin C

Troponin C (TnC) superfamily genes essentially possess five introns, the positions of all but the fourth being highly conserved. The fourth intron is frequently absent from protostomian invertebrate genes, such as calmodulin or TnC. We previously proposed that the common ancestor of TnC superfamily genes never possessed an intron corresponding to today's fourth introns, and that members of the superfamily independently gained a fourth intron in the evolutionary pathway of each lineage. In the present study, we isolated the TnC cDNA from the sandworm, Perinereis vancaurica tetradentata and determined its genomic structure. Sandworm TnC appears to exist as a single copy gene consisting of six exons and five introns. The positions of the first, second, third and fifth introns are identical to other TnCs, but that of the fourth intron is unique. This is in good agreement with the above-mentioned scheme, i.e. the gain of the fourth intron of sandworm TnC might have occurred within the annelid lineage after annelida/mollusca divergence.     

Early evolution and the origin of eukaryotes

Our understanding of evolutionary relationships in the eukaryotic world has been revolutionized by molecular systematics. Phylogenies based upon comparisons of rRNAs define five major eukaryotic assemblages plus a series of paraphyletic protist lineages. Comparison of conserved genes that were duplicated prior to the divergence of eubacteria, archaebacteria, and eukaryotes, positions the root of the universal tree within the eubacterial line of descent. In this review a novel model is presented which uses the rRNA and protein based phylogenies to describe the evolutionary origins of eukaryotes.     

Analysis of evolution of exon-intron structure of eukaryotic genes

The availability of multiple, complete eukaryotic genome sequences allows one to address many fundamental evolutionary questions on genome scale. One such important, long-standing problem is evolution of exon-intron structure of eukaryotic genes. Analysis of orthologous genes from completely sequenced genomes revealed numerous shared intron positions in orthologous genes from animals and plants and even between animals, plants and protists. The data on shared and lineage-specific intron positions were used as the starting point for evolutionary reconstruction with parsimony and maximum-likelihood approaches. Parsimony methods produce reconstructions with intron-rich ancestors but also infer lineage-specific, in many cases, high levels of intron loss and gain. Different probabilistic models gave opposite results, apparently depending on model parameters and assumptions, from domination of intron loss, with extremely intron-rich ancestors, to dramatic excess of gains, to the point of denying any true conservation of intron positions among deep eukaryotic lineages. Development of models with adequate, realistic parameters and assumptions seems to be crucial for obtaining more definitive estimates of intron gain and loss in different eukaryotic lineages. Many shared intron positions were detected in ancestral eukaryotic paralogues which evolved by duplication prior to the divergence of extant eukaryotic lineages. These findings indicate that numerous introns were present in eukaryotic genes already at the earliest stages of evolution of eukaryotes and are compatible with the hypothesis that the original, catastrophic intron invasion accompanied the emergence of the eukaryotic cells. Comparison of various features of old and younger introns starts shedding light on probable mechanisms of intron insertion, indicating that propagation of old introns is unlikely to be a major mechanism for origin of new ones. The existence and structure of ancestral protosplice sites were addressed by examining the context of introns inserted within codons that encode amino acids conserved in all eukaryotes and, accordingly, are not subject to selection for splicing efficiency. It was shown that introns indeed predominantly insert into or are fixed in specific protosplice sites which have the consensus sequence (A/C)AG|Gt.     

A survey of introns in three genes of rotifers

Here we report on the occurrence and position of introns found in three genes of rotifers. A region of the gene for the TATA-box binding protein was examined in three species of Bdelloidea and one of Monogononta. There are two introns in both copies of this gene present in each of the three bdelloids examined – one at a position where introns occur in other eukaryotes and the other at a novel position; the monogonont has no introns in the region examined. A region of the gene encoding the 82 kD heat shock protein was examined in 10 species, with every rotifer class represented. Introns were found in only two species, both bdelloids: one of the species has an intron in all three copies of the gene; the other has an intron in only one of the three copies. Both introns occur at novel positions. The gene for triosephosphate isomerase was examined in one bdelloid. Both copies of the gene in this species contain introns, all at conserved positions: one copy contains five introns, the other copy three. These observations demonstrate the presence of introns in bdelloid rotifers, some in conserved positions, others apparently newly arisen during bdelloid evolution.     

A putative ancestral actin gene present in a thermophilic eukaryote: novel combination of intron positions.

The gene encoding actin in the thermophilic fungus Thermomyces lanuginosus has been isolated and sequenced. It contains five introns, with three being at positions already known to be intron sites in actin genes from other eukaryotes. These three positions have not been found to occur simultaneously in any other organisms to date, suggesting that the actin gene in this fungus may more closely resemble an ancestral form of this highly conserved eukaryotic gene. The 5' flanking region of the gene contains a TATA-like sequence and two CCAAT motifs in positions almost identical to those in the yeast actin gene. Other features of the gene sequence, and possible adaptations to thermophily, are discussed.     

Conservation versus parallel gains in intron evolution

Orthologous genes from distant eukaryotic species, e.g. animals and plants, share up to 25–30% intron positions. However, the relative contributions of evolutionary conservation and parallel gain of new introns into this pattern remain unknown. Here, the extent of independent insertion of introns in the same sites (parallel gain) in orthologous genes from phylogenetically distant eukaryotes is assessed within the framework of the protosplice site model. It is shown that protosplice sites are no more conserved during evolution of eukaryotic gene sequences than random sites. Simulation of intron insertion into protosplice sites with the observed protosplice site frequencies and intron densities shows that parallel gain can account but for a small fraction (5–10%) of shared intron positions in distantly related species. Thus, the presence of numerous introns in the same positions in orthologous genes from distant eukaryotes, such as animals, fungi and plants, appears to reflect mostly bona fide evolutionary conservation.     

Conservation of the positions of metazoan introns from sponges to humans

Sponges (phylum Porifera) are the phylogenetic oldest Metazoa still extant. They can be considered as reference animals (Urmetazoa) for the understanding of the evolutionary processes resulting in the creation of Metazoa in general and also for the metazoan gene organization in particular. In the marine sponge Suberites domuncula, genes encoding p38 and JNK kinases contain nine and twelve introns, respectively. Eight introns in both genes share the same positions and the identical phases. One p38 intron slipped for six bases and the JNK gene has three more introns. However, the sequences of the introns are not conserved and the introns in JNK gene are generally much longer. Introns interrupt most of the conserved kinase subdomains I-XI and are found in all three phases (0, 1 and 2). We analyzed in details p38 and JNK genes from human, Caenorhabditis elegans and Drosophila melanogaster and found in most genes introns at the positions identical to those in sponge genes. The exceptions are two p38 genes from D. melanogaster that have lost all introns in the coding sequence. The positions of 11 introns in each of four human p38 genes are fully conserved and ten introns occupy identical positions as the introns in sponge p38 or JNK genes. The same is true for nine, out of ten introns in the human JNK-1 gene. The introns in human p38 and JNK genes are on average more than ten times longer than corresponding introns in sponges. It was proposed that yeast HOG1-like kinases (from i.e. Saccharomyces cerevisiae and Emericella nidulans) and metazoan p38 and JNK kinases are orthologues. p38 and JNK genes were created after the split from fungi by the duplication and diversification of the HOG1-like progenitor gene. Our results further support the common origin of p38 and JNK genes and speak in favor of a very early time of duplication. The ancestral gene contained at least ten introns, which are still present at the very conserved positions in p38 and JNK genes of extant animals. Four of these introns are present at the same positions in the HOG-like gene in the fungus E. nidulans. The others probably entered the ancestral gene after the split of fungi, but before the duplication of the gene and before the creation of the common, urmetazoan progenitor of all multicellular animals. A second gene coding for an immune molecule is described, the allograft inflammatory factor, which likewise showed a highly conserved exon/intron structure in S. domuncula and in human. These data show that the intron/exon borders are highly conserved in genes from sponges to human.     

Evolutionary dynamics of spliceosomal intron revealed by <Emphasis Type="Italic">in silico</Emphasis> analyses of the P-Type ATPase superfamily genes

It has been long debated whether spliceosomal introns originated in the common ancestor of eukaryotes and prokaryotes. In this study, we tested the possibility that extant introns were inherited from the common ancestor of eukaryotes and prokaryotes using in silico simulation. We first identified 21 intron positions that are shared among different families of the P-Type ATPase superfamily, some of which are known to have diverged before the separation of prokaryotes and eukaryotes. Theoretical estimates of the expected number of intron positions shared by different genes suggest that the introns at those 21 positions were inserted independently. There seems to be no intron that arose from before the diversification of the P-Type ATPase superfamily. Namely, the present introns were inserted after the separation of eukaryotes and prokaryotes.     

Ferritin gene organization: Differences between plants and animals suggest possible kingdom-specific selective constraints

Ferritin, a protein widespread in nature, concentrates iron ∼10¹¹–10¹²-fold above the solubility within a spherical shell of 24 subunits; it derives in plants and animals from a common ancestor (based on sequence) but displays a cytoplasmic location in animals compared to the plastid in contemporary plants. Ferritin gene regulation in plants and animals is altered by development, hormones, and excess iron; iron signals target DNA in plants but mRNA in animals. Evolution has thus conserved the two end points of ferritin gene expression, the physiological signals and the protein structure, while allowing some divergence of the genetic mechanisms. Comparison of ferritin gene organization in plants and animals, made possible by the cloning of a dicot (soybean) ferritin gene presented here and the recent cloning of two monocot (maize) ferritin genes, shows evolutionary divergence in ferritin gene organization between plants and animals but conservation among plants or among animals; divergence in the genetic mechanism for iron regulation is reflected by the absence in all three plant genes of the IRE, a highly conserved, noncoding sequence in vertebrate animal ferritin mRNA. In plant ferritin genes, the number of introns (n= 7) is higher than in animals (n= 3). Second, no intron positions are conserved when ferritin genes of plants and animals are compared, although all ferritin gene introns are in the coding region; within kingdoms, the intron positions in ferritin genes are conserved. Finally, secondary protein structure has no apparent relationship to intron/exon boundaries in plant ferritin genes, whereas in animal ferritin genes the correspondence is high. The structural differences in introns/exons among phylogenetically related ferritin coding sequences and the high conservation of the gene structure within plant or animal kingdoms suggest that kingdom-specific functional constraints may exist to maintain a particular intron/exon pattern within ferritin genes. In the case of plants, where ferritin gene intron placement is unrelated to triplet codons or protein structure, and where ferritin is targeted to the plastid, the selection pressure on gene organization may relate to RNA function and plastid/nuclear signaling. Received: 25 July 1995 / Accepted: 3 October 1995     

Complete sequence of a bovine type I cytokeratin gene: conserved and variable intron positions in genes of polypeptides of the same cytokeratin subfamily

The complete sequence of a bovine gene encoding an epidermal cytokeratin of mol. wt. 54 500 (No VIb) of the acidic (type I) subfamily is presented, including an extended 5' upstream region. The gene (4377 bp, seven introns) which codes for a representative of the glycine-rich subtype of cytokeratins of this subfamily, is compared with genes coding for: another subtype of type I cytokeratin; a basic (type II) cytokeratin gene; and vimentin, a representative of another intermediate filament (IF) protein class. The positions of the five introns located within the highly homologous alpha-helix-rich rod domain are identical or equivalent, i.e., within the same triplet, in the two cytokeratin I genes. Four of these intron positions are also identical with intron sites in the vimentin gene, and three of these intron positions are identical or similar in the type I and type II cytokeratin subfamilies. On the other hand, the gene organization of both type I cytokeratins differs from that of the type II cytokeratin in the rod region in five intron positions and in the introns located in the carboxy-terminal tail region, with the exception of one position at the rod-tail junction. Remarkably, the two type I cytokeratins also differ from each other in the positions of two introns located at and in the region coding for the hypervariable, carboxy-terminal portion. The introns and the 5' upstream regions of the cytokeratin VIb gene do not display notable sequence homologies with the other IF protein genes, but sequences identical with--or very similar to--certain viral and immunoglobulin enhancers have been identified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular evolution of eukaryotic genomes: hemiascomycetous yeast spliceosomal introns

As part of the exploratory sequencing program Génolevures, visual scrutinisation and bioinformatic tools were used to detect spliceosomal introns in seven hemiascomycetous yeast species. A total of 153 putative novel introns were identified. Introns are rare in yeast nuclear genes (<5% have an intron), mainly located at the 5′ end of ORFs, and not highly conserved in sequence. They all share a clear non-random vocabulary: conserved splice sites and conserved nucleotide contexts around splice sites. Homologues of metazoan snRNAs and putative homologues of SR splicing factors were identified, confirming that the spliceosomal machinery is highly conserved in eukaryotes. Several introns’ features were tested as possible markers for phylogenetic analysis. We found that intron sizes vary widely within each genome, and according to the phylogenetic position of the yeast species. The evolutionary origin of spliceosomal introns was examined by analysing the degree of conservation of intron positions in homologous yeast genes. Most introns appeared to exist in the last common ancestor of present day yeast species, and then to have been differentially lost during speciation. However, in some cases, it is difficult to exclude a possible sliding event affecting a pre-existing intron or a gain of a novel intron. Taken together, our results indicate that the origin of spliceosomal introns is complex within a given genome, and that present day introns may have resulted from a dynamic flux between intron conservation, intron loss and intron gain during the evolution of hemiascomycetous yeasts.     

Structural organization of lower marine nonvertebrate calmodulin genes.

The troponin C (TnC) superfamily genes generally possess five introns, and the positions where they are inserted are well conserved except for the fourth intron. Based on a structural comparison of TnC genes, we proposed that the common ancestor of TnC or TnC superfamily genes had no intron corresponding to the modern fourth intron, and therefore members of the superfamily have gained the fourth intron independently within each lineage. Here, we cloned calmodulin (CaM, one of the members of the TnC superfamily) cDNAs from two lower marine nonvertebrates, the sea anemone, Metridium senile, belonging to the Cnidaria, and the sponge, Halichondria okadai, belonging to the Porifera, and also determined their genomic organization. Chordate CaM genes generally possess five introns, but neither sea anemone nor sponge CaM has anything corresponding to the fourth intron of chordate CaMs, suggesting that the early metazoan CaM must have had only four introns. The modern fourth intron of chordate CaMs was acquired within the chordate lineage after nonvertebrate/chordate divergence. This notion concurs with our proposal explaining the evolution of the TnC superfamily genes.     

Nucleotide sequence of the triosephosphate isomerase gene from Aspergillus nidulans: implications for a differential loss of introns

A functional cDNA from Aspergillus nidulans encoding triosephosphate isomerase (TPI) was isolated by its ability to complement a tpi1 mutation in Saccharomyces cerevisiae. This cDNA was used to obtain the corresponding gene, tpiA. Alignment of the cDNA and genomic DNA nucleotide sequences indicated that tpiA contains five introns. The intron positions in the tpiA gene were compared with those in the TPI genes of human, chicken, and maize. One intron is present at an identical position in all four organisms, two other introns are located in similar positions in A. nidulans and maize, and the remaining two introns are unique to A. nidulans. These Aspergillus-specific introns are located in regions of the protein that were predicted to be interrupted by introns based on analysis of a Go plot of chicken TPI. These comparisons are discussed in relation to the evolution of introns within TPI genes.     

A canonical sequence organization at the 5'-end of the myosin heavy chain genes

The sequences encoding the 5'-ends of three chicken fast-white myosin heavy chain (MHC) genes have been determined. When compared with the sequences of two other MHC genes it is apparent that both the exon and intron positions are conserved. All exon sequences are highly conserved; there is absolute amino acid conservation in the second and third exons. In addition, while the first and third introns diverge among the genes, the second intron is highly conserved between the five. This intron contains a 24-bp sequence that is repeated twice in one of the introns and once in the other four. Analyses indicate that this sequence, which is partially homologous to 7SL RNA, appears to be largely restricted to the MHC gene family. Analysis of the 5'-flanking sequences show that while small homologies are present between some of the genes, they have extensively diverged in this region.     

Evidence for extensive recent intron transposition in closely related fungi

Though spliceosomal introns are a major structural component of most eukaryotic genes and intron density varies by more than three orders of magnitude among eukaryotes [1-3], the origins of introns are poorly understood, and only a few cases of unambiguous intron gain are known [4-8]. We utilized population genomic comparisons of three closely related fungi to identify crucial transitory phases of intron gain and loss. We found 74 intron positions showing intraspecific presence-absence polymorphisms (PAPs) for the entire intron. Population genetic analyses identified intron PAPs at different stages of fixation and showed that intron gain or loss was very recent. We found direct support for extensive intron transposition among unrelated genes. A substantial proportion of highly similar introns in the genome either were recently gained or showed a transient phase of intron PAP. We also identified an intron transfer among paralogous genes that created a new intron. Intron loss was due mainly to homologous recombination involving reverse-transcribed mRNA. The large number of intron positions in transient phases of either intron gain or loss shows that intron evolution is much faster than previously thought and provides an excellent model to study molecular mechanisms of intron gain.     

Molecular evolution of calmodulin-like domain protein kinases (CDPKs) in plants and protists

Many genes for calmodulin-like domain protein kinases (CDPKs) have been identified in plants and Alveolate protists. To study the molecular evolution of the CDPK gene family, we performed a phylogenetic analysis of CDPK genomic sequences. Analysis of introns supports the phylogenetic analysis; CDPK genes with similar intron/exon structure are grouped together on the phylogenetic tree. Conserved introns support a monophyletic origin for plant CDPKs, CDPK-related kinases, and phosphoenolpyruvate carboxylase kinases. Plant CDPKs divide into two major branches. Plant CDPK genes on one branch share common intron positions with protist CDPK genes. The introns shared between protist and plant CDPKs presumably originated before the divergence of plants from Alveolates. Additionally, the calmodulin-like domains of protist CDPKs have intron positions in common with animal and fungal calmodulin genes. These results, together with the presence of a highly conserved phase zero intron located precisely at the beginning of the calmodulin-like domain, suggest that the ancestral CDPK gene could have originated from the fusion of protein kinase and calmodulin genes facilitated by recombination of ancient introns. Received: 11 July 2000 / Accepted: 18 April 2001     

Analysis of Fungal Genomes Reveals Commonalities of Intron Gain or Loss and Functions in Intron-Poor Species

Previous evolutionary reconstructions have concluded that early eukaryotic ancestors including both the last common ancestor of eukaryotes and of all fungi had intron-rich genomes. By contrast, some extant eukaryotes have few introns, underscoring the complex histories of intron–exon structures, and raising the question as to why these few introns are retained. Here, we have used recently available fungal genomes to address a variety of questions related to intron evolution. Evolutionary reconstruction of intron presence and absence using 263 diverse fungal species supports the idea that massive intron reduction through intron loss has occurred in multiple clades. The intron densities estimated in various fungal ancestors differ from zero to 7.6 introns per 1 kb of protein-coding sequence. Massive intron loss has occurred not only in microsporidian parasites and saccharomycetous yeasts, but also in diverse smuts and allies. To investigate the roles of the remaining introns in highly-reduced species, we have searched for their special characteristics in eight intron-poor fungi. Notably, the introns of ribosome-associated genes RPL7 and NOG2 have conserved positions; both intron-containing genes encoding snoRNAs. Furthermore, both the proteins and snoRNAs are involved in ribosome biogenesis, suggesting that the expression of the protein-coding genes and noncoding snoRNAs may be functionally coordinated. Indeed, these introns are also conserved in three-quarters of fungi species. Our study shows that fungal introns have a complex evolutionary history and underappreciated roles in gene expression.     

A glimpse of a putative pre-intron phase of eukaryotic evolution

Comparison of the exon-intron structures of ancient eukaryotic paralogs reveals the absence of conserved intron positions in these genes. This is in contrast to the conservation of intron positions in orthologous genes from even the most evolutionarily distant eukaryotes and in more recent paralogs. The lack of conserved intron positions in ancient paralogs probably reflects the origination of these genes during the earliest phase of eukaryotic evolution, which was characterized by concomitant invasion of genes by group II self-splicing elements (which were to become introns in the future) and extensive duplication of genes.     

Tempo and Mode of Spliceosomal Intron Evolution in Actin of Foraminifera

Spliceosomal introns are present in almost all eukaryotic genes, yet little is known about their origin and turnover in the majority of eukaryotic phyla. There is no agreement whether most introns are ancestral and have been lost in some lineage or have been gained recently. We addressed this question by analyzing the spatial and temporal distribution of introns in actins of foraminifera, a group of testate protists whose exceptionally rich fossil record permits the calibration of molecular phylogenies to date intron origins. We identified 24 introns dispersed along the sequence of two foraminiferan actin paralogues and actin deviating proteins, an unconventional type of fast-evolving actin found in some foraminifera. Comparison of intron positions indicates that 20 of 24 introns are specific to foraminifera. Four introns shared between foraminifera and other eukaryotes were interpreted as parallel gains because they have been found only in single species belonging to phylogenetically distinctive lineages. Moreover, additional recent intron gain due to the transfer between the actin paralogues was observed in two cultured species. Based on a relaxed molecular clock timescale, we conclude that intron gains in actin took place throughout the evolution of foraminifera, with the oldest introns inserted between 550 and 500 million years ago and the youngest ones acquired less than 100 million years ago. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Debashish Bhattacharya]